This invention relates generally to an assay for identifying the presence and/or amount in a test sample of an antibody which is immunologically reactive with a hepatitis C viral antigen and more specifically, to an assay for detecting a complex of an antibody and a polypeptide having at least one epitope of a hepatitis C viral antigen.
Acute viral hepatitis is clinically diagnosed by a well-defined set of patient symptoms, including jaundice, hepatic tenderness, and an increase in the serum levels of alanine aminotransferase and aspartate aminotransferase. Additional serological immunoassays are generally performed to diagnose the specific type of viral causative agent. Historically, patients presenting clinical hepatitis symptoms and not otherwise infected by hepatitis A, hepatitis B, Epstein-Barr, or cytomegalovirus were clinically diagnosed as having non-A, non-B hepatitis (NANBH) by default. The disease may result in chronic liver damage.
Each of the well-known, immunologically characterized hepatitis-inducing viruses, hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis D virus (HDV) belongs to a separate family of viruses and has a distinctive viral organization, protein structure and mode of replication.
Attempts to identify the NANBH virus by virtue of genomic similarity to one of the known hepatitis viruses have failed, suggesting that NANBH has a distinct organization and structure. Fowler et al., J. Med. Virol. 12:205-213 (1983) and Weiner et al., J. Med. Virol. 21:239-247 (1987).
Progress in developing assays to detect antibodies specific for NANBH has been particularly hampered by difficulties in correctly identifying antigens associated with NANBH. See, for example, J. Wands et al., U.S. Pat. No. 4,870,076, Wands et al., Proc. Nat""l. Acad. Sci. 83:6608-6612 (1986), Ohori et al., J. Med. Virol. 12:161-178 (1983), Bradley et al., Proc. Nat""l. Acad. Sci. 84:6277-6281 (1987), T. Akatsuka et al., J. Med. Virol. 20:43-56 (1986), B. Seto et al., U.S. patent application Ser. No. 07/234,641 (available from U.S. Department of Commerce National Technical Information Service, Springfield, Va. No. 89138168), K. Takahashi et al., European Patent Application No. 0 293 274, published Nov. 30, 1988, and R. Seelig et al., PCT Application PCT/EP88/00123.
Recently, another hepatitis-inducing virus has been unequivocally identified as hepatitis C virus (HCV) by M. Houghton et al., European Patent Application publication number 0 318 216, May 31, 1989. Related papers describing this virus include G. Kuo et al., Science 244:359-361 (1989) and Q. Choo et al., Science 244:362-364 (1989). M. Houghton et al. reported isolating cDNA sequences from HCV which encode antigens which react immunologically with antibodies present in patients infected with NANBH, thus establishing that HCV is the viral agent causing NANBH.
The CDNA sequences associated with HCV were isolated from a cDNA library prepared from the RNA obtained from pooled serum from a chimpanzee with chronic HCV infection. The cDNA library contained cDNA sequences of approximate mean size of about 200 base pairs. The cDNA library was screened for encoded epitopes expressed in clones that could bind to antibodies in sera from patients who had previously experienced NANBH.
In the European Patent Application, M. Houghton et al. also described the preparation of several superoxide dismutase fusion polypeptides (SOD) and the use of these SOD fusion polypeptides to develop an HCV screening assay. The most complex SOD fusion polypeptide described in the European Patent Application, designated C100-3, was described as containing 154 amino acids of human SOD at the amino-terminus, 5 amino acid residues derived from the expression of a synthetic DNA adapter containing a restriction site, EcoRI, 363 amino acids derived from the expression of a cloned HCV cDNA fragment, and 5 carboxy terminal amino acids derived from an MS2 cloning vector nucleotide sequence. The DNA sequence encoding this polypeptide was transformed into yeast cells using a plasmid. The transformed cells were cultured and expressed a 54,000 molecular weight polypeptide which was purified to about 80% purity by differential extraction.
Other SOD fusion polypeptides designated SOD-NANB5-1-1 and SOD-NANBH81 were expressed in recombinant bacteria. The E. coli fusion polypeptides were purified by differential extraction and by chromatography using anion and cation exchange columns. The purification procedures were able to produce SOD-NANB5-1-1 as about 80% pure and NANBH81 as about 50% pure.
The recombinant SOD fusion polypeptides described by M. Houghton et al. were coated on microtiter wells or polystyrene beads and used to assay serum samples. Briefly, coated microtiter wells were incubated with a sample in a diluent. After incubation, the microtiter wells were washed and then developed using either a radioactively labelled sheep anti-human antibody or a mouse anti-human IgG-HRP (horseradish peroxidase) conjugate. These assays were used to detect both post acute phase and chronic phase of HCV infection. Due to the preparative methods, assay specificity required adding yeast or E. coli extracts to the samples in order to prevent undesired immunological reactions with any yeast or E. coli antibodies present in serum samples.
Ortho Diagnostics Systems Inc. have developed a research immunoenzyme assay to detect antibodies to HCV antigens. The Ortho assay procedure is a three-stage test for serum/plasma carried out in a microwell coated with the recombinant yeast/hepatitis C virus SOD fusion polypeptide C100-3.
In the first stage, a test specimen is diluted directly in the test well and incubated for a specified length of time. If antibodies to HCV antigens are present in the specimen, antigen-antibody complexes will be formed on the microwell surface. If no antibodies are present, complexes will not be formed, and the unbound serum or plasma proteins will be removed in a washing step.
In the second stage, anti-human IgG murine monoclonal antibody horseradish peroxidase conjugate is added to the microwell. The conjugate binds specifically to the antibody portion of the antigen-antibody complexes. If antigen-antibody complexes are not present, the unbound conjugate will also be removed by a washing step.
In the third stage, an enzyme detection system composed of o-phenylenediamine 2 HCl (OPD) and hydrogen peroxide is added to the test well. If bound conjugate is present, the OPD will be oxidized, resulting in a colored end product. After formation of the colored end product, dilute sulfuric acid is added to the microwell to stop the color-forming detection reaction.
The intensity of the colored end product is measured with a microwell reader. The assay may be used to screen patient serum and plasma.
It is established that HCV may be transmitted by contaminated blood and blood products. In transfusion patients, as many as 10% will suffer from post-transfusion hepatitis. Of those, approximately 90% are the result of infections diagnosed as HCV. The prevention of transmission of HCV by blood and blood products requires reliable, sensitive and specific diagnosis and prognostic tools to identify HCV carriers as well as contaminated blood and blood products. Thus, there exists a need for an HCV assay which uses reliable and efficient reagents and methods to accurately detect the presence of HCV antibodies in samples.
The present invention provides an improved assay for detecting the presence of an antibody to an HCV antigen in a test sample by contacting the sample with a polypeptide containing at least one epitope of an HCV antigen, wherein the polypeptides are selected from the group consisting of p380-JH1, p-380.LG, p380-J and p408.
One assay format according to the invention provides a confirmatory assay for unequivocally identifying the presence of an antibody that is immunologically reactive with an HCV antigen. Briefly, a fluid sample is used to prepare first and second aliquots. The aliquots then are contacted with at least two polypeptides duplicative of a continuous amino acid sequence putatively contained in proteins expressed by clones containing HCV cDNA sequences containing at least one epitope of an HCV antigen under conditions suitable for complexing the antibody with the polypeptide. Finally, the antibody-antigen complex is detected. The first aliquot and the second aliquot are contacted with at least one polypeptide selected from the group consisting of p380-JH1, p-380.LG, p380-J and p408J, with the proviso that the polypeptide(s) used for the first aliquot are not used for the second aliquot.
Another assay format provides an assay for identifying the presence of an antibody that is immunologically reactive with an HCV antigen in a fluid sample comprising contacting the sample with a solid phase having bound thereto at least one polypeptide selected from the group consisting of p380JH1, p380J, p380.LG and p408J containing at least one epitope of an HCV antigen, under conditions suitable for complexing the antibody with the polypeptide and detecting the antibody-polypeptide complex.
Another assay format provides an assay for identifying the presence of an antibody that is immunologically reactive with an HCV antigen in a fluid sample comprising contacting the sample with a polypeptide selected from the group consisting of p380JH1, p380J, p380.LG and p408J containing at least one epitope of an HCV antigen under conditions suitable for complexing the antibody with the polypeptide and detecting the antibody-polypeptide complex as an indication of chronic HCV infection.
Another assay format provides an immunodot assay for identifying the presence of an antibody that is immunologically reactive with an HCV antigen by concurrently contacting a sample with at least two polypeptides selected from the group consisting of p380JH1, p380J, p380.LG and p408J each containing distinct epitopes of an HCV antigen under conditions suitable for complexing the antibody with at least one of the polypeptides and detecting the antibody-polypeptide complex by reacting the complex with color-producing reagents.
Another assay format provides a competition assay directed to the confirmation that positive results are not false by identifying the presence of an antibody that is immunologically reactive with an HCV antigen in a fluid sample where the sample is used to prepare first and second immunologically equivalent aliquots. The first aliquot is contacted with a solid support containing a bound polypeptide selected from the group consisting of p380JH1, p380J, p380.LG and p408J which contains at least one epitope of an HCV antigen under conditions suitable for complexing with the antibody to form a detectable antibody-polypeptide complex and the second aliquot is first contacted with unbound polypeptide selected from the group consisting of p380JH1, p380J, p380.LG and p408J and then contacted with the same solid support containing bound polypeptide selected from the group consisting of p380JH1, p380J, p380.LG and p408J.
In all of the assays, the sample, especially serum or plasma, preferably is diluted before contacting the polypeptide absorbed on a solid support. However, it is contemplated that when testing some test samples, an undiluted or even concentrated sample may be preferred. Samples may be obtained from different biological samples such as whole blood, serum, plasma, cerebrospinal fluid, urine, and lymphocytes or cell culture supernatants. Solid support materials may include cellulose materials, such as paper and nitrocellulose, natural and synthetic polymeric materials, such as polyacrylamide, polystyrene, and cotton, silicon chips, porous gels such as silica gel, agarose, dextran and gelatin, particles which are capable of forming a charge such as those used for ion capture assays and inorganic materials such as deactivated alumina, magnesium sulfate and glass. Suitable solid support materials may be used in assays in a variety of well known physical configurations, including microtiter wells, test tubes, beads, strips, membranes, and microparticles, either magnetic or non-magnetic. A preferred solid support for a non-immunodot assay is a polystyrene bead. A preferred solid support for an immunodot blot assay is nitrocellulose.
Suitable methods and reagents for detecting an antibody-antigen complex in an assay of the present invention are commercially available or known in the relevant art. Representative methods may employ detection reagents such an enzymatic, radioisotopic, fluorescent, luminescent or chemiluminescent reagents. These reagents may be used to prepare hapten-labelled anti-hapten detection systems according to known procedures, for example, a biotin-labelled anti-biotin system may be used to detect an antibody-antigen complex.
The present invention also encompasses assay kits including polypeptides which contain at least one epitope of an HCV antigen bound to a solid support as well as needed sample preparation reagents, wash reagents, detection reagents and signal producing reagents.
Other aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the following detailed description which provides illustrations of the invention in its presently preferred embodiments.